A number of aging theories predict that there is a relationship between somatic mutations and aging processes, but few systems exist to test this hypothesis at a specific locus. This proposal describes the construction of a model system which will ultimately allow for: 1) a quantitative assessment of the frequency of somatic mutations as a function of age, 2) a determination of the mechanisms of somatic cell mu-action in vivo, and 3) a qualitative assessment of changes in the types of mutations in somatic cells during growth and development. To accomplish this, a cohort of mice will be constructed with a heterozygous deficiency at a defined locus and a primary cloning assay will be used to identify and analyze those cells which have undergone a mutation leading to the homozygous deficient state. The construction of mice with a specific heterozygous deficiency requires a mutation at a selectable locus and a means to introduce this mutation into a mouse population. The autosomal purine salvage locus adenine phosphoribosyltransferase (APRT) is chosen for this study because: it is selectable and dispensable in mammalian cells and a DNA probe for the mouse gene is available. To construct mice with a heterozygous deficiency at the APRT locus, the mouse embryonal stem cell system will be used. These cultured stem cells are capable of differentiating into numerous cell types under the appropriate stimulus. Most dramatically, they can be injected into a developing mouse blastocyst and will participate in formation of a variety of organs. The resultant mouse, termed a chimera, is therefore partially derived from a cell type which was maintained in vitro. By selecting for an embryonal cell with a homozygous deficiency for APRT, it will be possible to construct a chimeric mouse with this deficiency. Further, by breeding functional germ line chimeras with wild-type mice, it will be possible to obtain F1 progeny with heterozygous deficiencies for APRT. These mice will then be used for long-term studies to determine if the frequency and spectrum of somatic cell mutations at a specific autosomal locus are altered as a function of age.